Interaction of Polyphenols with Proteins: Binding of (-)-Epigallocatechin Gallate to Serum Albumin, Estimated by Induced Circular Dichroism

ExperimentalReagents and Polyphenol HSA (cat.No. A3782) and BSA (cat.No. A0281) used in this study were of the fatty acid free grade (Ն99%).These two proteins, phenylbutazone (PB), tolbutamide (TB), ibuprofen (IP), and digitoxin (DG) were purchased from Sigma (St. Louis, MO, U.S.A.).Diazepam (DP) and ethacrynic acid (EA) were purchased from Wako (Osaka, Japan) and Tokyo Kasei (Tokyo, Japan), respectively.EGCG was isolated from leaves of Thea sinensis L. (ϭCamellia sinensis O. KUNTZE), as reported previously.17) CD Spectral Measurements CD spectra between 355 and 245 nm were recorded on a JASCO J-720W spectropolarimeter (Tokyo, Japan), using a 0.1-cm path-length cell at 298-301 K for the experiments using HSA, or at 295-297 K for the BSA experiments.Time constant, scan speed, resolution, and sensitivity were set at 1 s, 100 nm/min, 1.0 nm and 5 mdeg, respectively.Twelve scanned spectra were accumulated for the spectral measurements.The changes in the amplitudes of the induced Cotton effects were measured when the resolution was set at 0.5 nm, and the data from 32 scans were given as the means of the three points around the indicated wavelength.All experiments were performed in the solution of 0.1 M K 2 HPO 4 -KH 2 PO 4 buffer (pH 7.0), and the reagent (EGCG and drugs) were soluble in the buffer at the concentrations used.The final concentration of HSA was set 224Vol. 57, No.